High-throughput mapping of antigen specificity to B-cell-receptor sequence for characterizing antibody responses in HIV-vaccinated and infected individuals

B 细胞受体序列抗原特异性的高通量图谱，用于表征 HIV 疫苗接种者和感染者的抗体反应

• 批准号: 10478203
• 负责人: Ivelin Georgiev
• 金额: $ 85.71万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-02 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10478203
• 关键词: Address; Affect; Antibodies; Antibody Repertoire; Antibody Response; Antibody Therapy; Antigen Targeting; Antigens; Atlases; B-Cell Antigen Receptor; B-Lymphocyte Epitopes; B-Lymphocytes; Bar Codes; Benchmarking; Binding Sites; Biological Assay; Cell Line; Cells; Characteristics; Collection; Complex; DNA; Epitope Mapping; Epitopes; Event; Generations; Goals; HIV; HIV Infections; HIV Vaccine Trials Network; HIV-1; HIV-1 vaccine; Human; Immune system; Immunodominant Epitopes; Individual; Infection; Knock-out; Lead; Libraries; Light; Link; Maps; Measures; Monoclonal Antibodies; Mutation; Peptides; Performance; Polysaccharides; Prevention; Process; Recovery; Resolution; Sampling; Sequence Determination; Specificity; Technology; Time; Vaccinated; Vaccination; Vaccine Design; Vaccines; Validation; analysis pipeline; antigen binding; cell determination; co-infection; cohort; experimental study; next generation sequencing; novel; pathogen; prevent; response; screening; technology development; tool; vaccine candidate; vaccine-induced antibodies

Project Summary. The search for an effective HIV-1 vaccine remains a top priority, and a deeper understanding of how the immune system recognizes HIV-1 can help inform vaccine design. Lately, much effort has focused on understanding antibody responses to HIV-1 infection and vaccination, since antibodies have proven useful in therapy and prevention, and as templates for antibody-specific vaccine design. While antibody responses to HIV-1 are polyclonal and complex, advances in next-generation sequencing (NGS) technologies enable us to see such polyclonal responses at an unprecedented resolution, as a collection of individual monoclonal antibody sequences. Sequence identification is typically followed by functional antibody characterization, a primary component of which is the mapping of antigen/epitope specificity. A major challenge with the standard antibody analysis pipeline is that the sequence identification and functional characterization processes for antibodies are generally decoupled. This prevents truly high- throughput mapping of antibody-antigen specificity, providing only limited information for a small subset of selected antibody sequences from any given sample. To address this challenge, here we propose to develop a technology that, for a given sample, will enable the mapping of antibody sequence to antigen specificity from a single high-throughput experiment. The technology, LIBRA-seq (LInking B-cell Receptor to Antigen specificity through sequencing), involves physically mixing a B-cell sample with a (theoretically unlimited) pool of barcoded antigens, thus enabling the simultaneous recovery of: (i) paired heavy-light chain BCR sequences and (ii) antigen specificity for a given B cell. In particular, this technology development project will broadly focus on two specific aims: In Specific Aim 1, we will evaluate the effect of different antigen barcoding strategies and other assay variables on LIBRA-seq accuracy and performance. The goal in this aim is to optimize the LIBRA- seq ability to accurately detect BCR sequence and antigen specificity from a sequencing experiment. In Specific Aim 2, we will aim to simultaneously map the target epitope of a given HIV-specific B cell, by screening a cocktail of antigens with epitope-knockout mutations along with the wildtype antigens. These efforts will not only lead to the identification of HIV-specific B cells, but will also provide residue-level information about the specific epitope target on the antigen from the same high-throughput experiment. Ultimately, for a given infection or vaccination sample, the LIBRA-seq technology will provide the ability to recover antibody sequence and antigen specificity for tens to hundreds of thousands of B cells at the single- cell level. To demonstrate the utility of LIBRA-seq, we will characterize samples from HIV-1 infection and vaccination cohorts. More generally, LIBRA-seq will be an integral tool for efficient and accurate B-cell analysis, with the potential for broad impact on the fields of vaccine and antibody discovery not only for HIV-1 but also for a wide range of other pathogens of biomedical significance.

项目摘要。寻找有效的 HIV-1 疫苗仍然是当务之急，并且需要更深入的研究 了解免疫系统如何识别 HIV-1 有助于为疫苗设计提供信息。最近，很多 工作重点是了解抗体对 HIV-1 感染和疫苗接种的反应，因为抗体 已被证明可用于治疗和预防，并可作为抗体特异性疫苗设计的模板。尽管 HIV-1 抗体反应是多克隆且复杂的，下一代测序 (NGS) 取得进展 技术使我们能够以前所未有的分辨率看到这种多克隆反应，作为 单独的单克隆抗体序列。序列鉴定之后通常是功能性抗体 表征，其主要组成部分是抗原/表位特异性的映射。 标准抗体分析流程的一个主要挑战是序列识别和 抗体的功能表征过程通常是解耦的。这可以防止真正的高 抗体-抗原特异性的通量图，仅提供一小部分的有限信息 从任何给定样品中选择抗体序列。为了应对这一挑战，我们建议开发一个 对于给定的样品，该技术将能够将抗体序列映射到抗原特异性 单一高通量实验。 LIBRA-seq 技术（将 B 细胞受体与抗原特异性连接起来） 通过测序），涉及将 B 细胞样本与（理论上无限的）池进行物理混合 条形码抗原，从而能够同时恢复：（i）配对的重链-轻链 BCR 序列 (ii) 对给定 B 细胞的抗原特异性。特别是，该技术开发项目将广泛关注 关于两个具体目标：在具体目标 1 中，我们将评估不同抗原条形码策略的效果和 其他影响 LIBRA-seq 准确性和性能的分析变量。这一目标的目标是优化 LIBRA- seq 能够通过测序实验准确检测 BCR 序列和抗原特异性。在 具体目标 2，我们的目标是同时绘制给定 HIV 特异性 B 细胞的目标表位，方法是 筛选具有表位敲除突变的抗原混合物以及野生型抗原。这些 努力不仅会导致 HIV 特异性 B 细胞的鉴定，而且还将提供残留水平 来自同一高通量实验的有关抗原上特定表位靶标的信息。 最终，对于给定的感染或疫苗接种样本，LIBRA-seq 技术将提供以下能力： 恢复数万至数十万个 B 细胞的抗体序列和抗原特异性 细胞水平。为了证明 LIBRA-seq 的实用性，我们将对 HIV-1 感染样本进行表征，并 疫苗接种队列。更一般地说，LIBRA-seq 将成为高效、准确的 B 细胞的不可或缺的工具 分析，不仅针对 HIV-1，还可能对疫苗和抗体发现领域产生广泛影响 而且对于广泛的其他病原体也具有生物医学意义。